Conducting airway epithelium is one of the target tissues of ozone injury. The nature of this injury is complex due to the interactions between ozone and a large number of cellular components. In order to understand the nature of the ozone injury and its toxicity at the cellular level, we propose to establish an in vitro cell culture model for the ozone exposure. The in vitro model in some way should reflect the in vivo situation that most airway epithelial cells are facing the lumen and withdraw their nutrients and hormones/growth factors from the interstitial layer. We have developed an organotypic culture system, namely the Whitcutt chamber, for this purpose. Preliminary studies with this chamber demonstrate that cultured cells grown under the biphasic condition maintain the polarity of differentiation with all the differentiated features in the apical region (facing the air). In this proposal, we will use this system to examine the ozone toxicity on airway epithelial cells. We will develop an in vitro model of "elevated ozone resistance". In addition to these developments, we will test the following two hypothesis: 1) Ozone induces a response similar to that of heat shock. This response includes the synthesis of heat shock proteins (HSPs). 2) HSPs or HSP-like proteins are partly involved in the phenomenon of "elevated ozone resistance". In order to approach these hypotheses, cellular proteins before and after ozone exposure will be labeled and analyzed in SDS-PAGE. HSP-like proteins will be identified by the Western blot. The nature of HSPs synthesis will be further determined in vivo in ozone-exposed tissues by the immunocytochemical method. These interdisciplinary approaches will help not only to have a better understanding of the nature of ozone injury but to devise a protocol of prevention of ozone toxicity.